Skeletal muscle comprises about 40% of body weight and contains 50–75% of all proteins in the human body. Therefore, aside from its role in converting chemical energy into mechanical energy for movement and posture, muscle is also essential for the regulation of amino acid metabolism (e.g., oxidizing the branched chain amino acids and producing alanine and glutamine as gluconeogenic substrates and fuels for other tissues). In addition, muscle is a store of energy and nitrogen, which becomes vital for supply of fuel for the brain and the immune system, and substrate for wound healing during malnutrition, starvation, injury, and disease. The mechanisms that regulate the maintenance of the muscle mass are complex. As well as long term genetic influences, skeletal muscle can be influenced by a variety of factors including physical activity, nutrition (primarily amino acids), hormones (insulin, testosterone, growth hormone (GH)/insulin-like growth factor-1 (IGF-1), cortisol), and disease or trauma. Moreover, the maintenance of body protein mass is critical not only for remaining physically independent, but also, more importantly, for survival. In fact, the loss of approximately 30% of the body proteins results in impaired respiration and circulation due to muscle weakness and reduced immune function due to lack of nutrients, eventually resulting in death.
The turnover of body proteins, which is comprised of the simultaneous and energetic processes of protein synthesis and protein breakdown, may account for ∼20% of resting energy expenditure (Fig. 1). In fact, quantitative estimates suggest that about 1–2% of all skeletal muscle is synthesized and broken down daily. Even though skeletal muscle protein turnover is relatively slow in comparison with other tissues, the mass of skeletal muscle means that it accounts for a large part of whole-body protein turnover, about 30–50%, depending on age and dietary conditions. The ability of skeletal muscle to adapt rapidly to physiological variation (e.g., physical activity of various types, dietary change in quality and quantity, oxidative damage, etc.) must involve modulation of muscle protein synthesis and breakdown. Both synthesis and breakdown are controlled by cellular mechanisms that include the activation of gene transcription, initiation of protein synthesis, and several proteolytic enzyme pathways. As a result of work carried out over the last 20 yrs, we are now beginning to understand the relative importance of these pathways and how they are regulated in human skeletal muscle.
Muscle protein anabolism occurs whenever the rate of muscle protein synthesis is greater than the rate of muscle protein breakdown, resulting in a net accretion of muscle protein over time. In healthy adults, muscle proteins are alternatively being synthesized (net deposition of amino acids) or degraded (breakdown), which can result in a net release of amino acids. A state of net synthesis or breakdown depends primarily upon the state of feeding (see below), and when protein intake is adequate, in the absence of any other stimulus, there is no net growth or loss. However, a repeated observation is that resistance exercise can dramatically stimulate muscle cell hypertrophy. Therefore, the focus of this review will be on the mechanisms of how resistance exercise, nutritional provision, and the combination of the two results in muscle growth (i.e., anabolism).
Resistance exercise can induce profound muscular hypertrophy, which is responsible for a variable degree of strength gain. Obviously, hypertrophy or a net gain in muscle mass is due to an extended period where protein synthesis has exceeded protein breakdown. The time course of hypertrophy is relatively slow, generally taking weeks or months to be apparent. This prolonged time course for hypertrophy may be due, to some degree, to the limited sensitivity of techniques available to measure hypertrophy, but is primarily a reflection of the slow rate of turnover of muscle proteins. When considered in the absence of other processes, a protein fractional synthetic rate (FSR) in skeletal muscle of as low as 1%·d−1, should yield fiber hypertrophy in far less than 8 wk (assuming an adequate sample size). Of course, what is not taken into account when regarding only muscle protein synthesis is the opposing rate of skeletal muscle protein breakdown (Fig. 1). Moreover, as has been demonstrated a number of times, the rate of synthesis and breakdown seem to be quite closely linked (8,9). In fact, it is the balance between rates of protein synthesis and breakdown that determines net balance and net protein gains (i.e., hypertrophy). This axiom is true regardless of the underlying mechanism(s) of hypertrophy.
Resistance exercise is a potent stimulator of muscle protein synthesis (7–9). Perhaps more impressive is the long-lasting effect that resistance exercise has on muscle protein FSR (Fig. 2) (8). Several studies have attempted to delineate the time course of the resistance-exercise–induced increase in muscle protein FSR and have reached somewhat different conclusions. The data from these studies is summarized in Figure 2, which shows that a single isolated bout of resistance exercise stimulates muscle protein synthesis very rapidly (within 2 to 4 h). Moreover, that the stimulation of protein synthesis lasts for at least 24 h (7), and in one study for 48 h (8), at which time protein synthesis was still elevated above preexercise levels, suggests that the elevation of protein synthesis may last even longer than 48 h. However, the data in Figure 2 illustrate that there is not complete concordance over the exact time course. The results from the data of MacDougall et al. (7) indicate that 24 h after a bout of resistance exercise, muscle FSR was still 109% above baseline, whereas at 4 h the increase was only 50% above baseline. By adding a third group of subjects, the authors concluded that by 36 h postexercise, muscle protein FSR had returned to baseline (Fig. 2) (7). However, the subjects in this study (7) were trained resistance athletes and different groups of subjects were studied at each time point postexercise to construct the overall time course. From the perspective of how training affects muscle protein synthesis, as will be discussed subsequently, training substantially attenuates the response of muscle protein synthesis. In contrast, Phillips et al. (8) studied untrained subjects and used a repeated measures design, which may have explained the differing time course of FSR than that observed by MacDougall et al. (7) (see Fig. 2). Hence, the difference between the time course of protein synthesis after an isolated bout of resistance exercise reported by MacDougall et al. (7) and that reported by Phillips et al. (8) may be due to the training status of the subjects.
Until the appearance of recent studies, the increase in mixed muscle protein synthesis (MMP) observed after resistance exercise could not conclusively be established as the result of an increase in myofibrillar protein synthesis per se (7–9). Although a resistance-exercise–induced increase in myofibrillar protein synthesis may seem intuitive, because resistance exercise does eventually result in hypertrophy and strength gains as a result of the accumulation of force-producing myofibrillar proteins, recent studies have now established that this is in fact the case (2,5). These findings clearly establish that the increase in mixed muscle protein synthesis observed after exercise (7–9) is predominantly due to myofibrillar protein synthesis, most likely myosin heavy chain and actin (2,5). This conclusion gains further weight when one considers the fact that myofibrillar proteins comprise the majority of proteins in muscle; hence, it would be difficult to envision that changes in mixed muscle protein synthesis after resistance exercise would disproportionately stimulate the synthesis of either sarcoplasmic or mitochondrial protein fractions.
The successful adaptation of physiological systems to a stressful stimulus is to reduce the magnitude of the stress response. Hence, data from studies that have examined the response of repetitive bouts of resistance exercise (i.e., training), because exercise is a stressor, have shown that training diminishes the response of MMP FSR to a given exercise load (9). This finding was tested in a cross-sectional design in which a group of resistance-trained males and females performed exercise at the same relative intensity as a group of untrained males and females. The results showed that at the same relative intensity the group of trained subjects had a smaller increase in MMP FSR (9). In contrast, the untrained group had large increases in both MMP FSR and fractional breakdown rate (FBR). As is incumbent with all cross-sectional designs there is the possibility that preexisting genetically determined differences, other than training status, could be responsible for the findings. Hence, using a longitudinal design we have recently examined the response to an acute bout of exercise before and after 8 wk of resistance training in a group of novices. These results, in which subjects were examined at the same absolute workload pre- and post-training, showed that training reduced the exercise-induced rise in MMP FSR (10). Choosing to test subjects at the same absolute load pre- and post-training is the only way to determine the effect of the training program on muscle protein turnover. Hence, using the same relative intensity pre- versus post-training means that both absolute strength, due to training, and intensity (i.e., ATP turnover) would have been manipulated, which means that the results obtained do not answer how “training”per se affected protein turnover. However, two studies have confirmed that training does reduce the synthesis of MMP in response to an acute bout of exercise at both the same relative (9) and absolute workload (10).
Interestingly, it was shown that resistance training resulted in an elevated resting rate of MMP FSR (9), although this did not reach statistical significance; however, this was recently confirmed in our longitudinal training study (10). In the absence of data on how training affected FBR, one might have predicted that training, by elevating resting FSR, would have theoretically elevated protein requirements. The fact that MMP FBR rose in concert with FSR meant that net balance was unchanged, but that turnover was markedly elevated after training (10). This training-induced elevation in muscle protein turnover (i.e., FSR and FBR) means that the processes of protein synthesis and degradation must have somehow been “amplified,” which is indicative of an environment in which more rapid protein remodeling is occurring (10).
Although there is no doubt that regular resistance exercise will change the expression of various genes, it is not understood how such transcriptional changes are regulated by exercise. Moreover, it would appear that a large proportion of the changes in muscle protein turnover that occur after resistance exercise are posttranscriptional, largely due to increased translational efficiency. This conclusion is reached based on the rapidity of the response in muscle protein synthesis that is observed (i.e., within 3–4 h after completion of a resistance exercise bout), which is unlikely to allow for substantial changes in gene expression. In support of this thesis, a variety of translational regulatory factors, including eukaryotic initiation factors (eIF) have been studied in rodent muscle. Interestingly, resistive exercise provides a stimulus to activate eIF2B, but has less of an effect on eIF4E 4G complex formation (work from Peter Farrell’s laboratory). Another potential activator of protein synthesis initiation is the p70-kDa S6 protein kinase (p70S6K), which stimulates the initiation of protein synthesis once the S6 protein of the ribosome, is phosphorylated. Baar and Esser (1) showed that the activity (phosphorylation) of p70S6K correlated well with the “training-induced” increase in skeletal muscle mass brought about by high force resistive contractions, in rats. These results (1) demonstrate the importance of the activation of this protein kinase in potentially governing the hypertrophic response to resistive exercise.
Rennie and colleagues performed the first study of the effect of feeding on human muscle protein synthesis in 1982 and this study was followed by a number of other studies, from several groups, using both incorporation of isotope labeled amino acids or tracer limb exchange methods (11). In summary, from these studies we can conclude that: 1) muscle protein synthesis increases between 30–100% in response to oral or intravenous feeding (11,14); 2) a major component of this effect is due to the stimulation of protein synthesis by amino acids; 3) the effect of insulin on muscle protein synthesis is dependent on the availability of amino acids; and 4) the major component of the anabolic response is a stimulation of protein synthesis rather than a diminution of protein breakdown. Thus, feeding causes a substantial increase in muscle protein synthesis and a lesser inhibition of muscle protein breakdown, the net result being an increase in muscle protein accretion. The anabolic response of feeding appears to be transient and within a few hours after the meal, or after an overnight fast, these two processes are reversed (i.e., breakdown > synthesis, Fig. 3).
There are surprisingly few data in humans to indicate exactly how feeding results in acute (1–3 h) increases in muscle anabolism, except that gene expression is not involved (though it is likely to be in longer-term responses). Amino acids, in particular essential amino acids, can directly stimulate human muscle protein synthesis including the main intracellular components (myofibrillar, sarcoplasmic, and mitochondrial fractions), and they do so in a dose-dependent manner (4). However, the mechanism(s) of how a muscle cell senses an increased availability of amino acids and signals to the protein synthetic and degradative apparatus is far from being understood although progress is being made by analogy to data obtained from animal muscle. There appear to be several intracellular signaling pathways that sense the amino acids, transmit the signal to the cell interior, and activate the initiation of translation of proteins from mRNA (6).
During a normal feeding episode, amino acid and insulin concentrations increase and can promote muscle protein synthesis through distinct pathways. For example, it is known from work in animals that essential amino acids can activate the mammalian target of rapamycin (mTOR), which then promotes the phosphorylation of eIF4E-BP1 inducing the binding protein to release eIF4E (6). The active complex of eIF4E bound with eIF4G can stimulate the initiation of translation of specific mRNAs associated with the initiation process. Phosphorylation of eIF4E-BP1 may also stimulate translation of general or global protein synthesis. Very little research of these signaling pathways have been reported in human skeletal muscle and much research is needed in humans to elucidate specific mechanisms that regulate muscle protein turnover.
The role of insulin is less straightforward. Both amino acids and carbohydrate in a mixed meal will stimulate insulin secretion (12,15). It is incontrovertible that insulin can stimulate muscle anabolism by inhibiting muscle protein breakdown; however, the question of the effect of insulin on muscle protein synthesis is less clear and is somewhat controversial. Our view, which we believe is the most widely accepted, is that insulin acts at low concentrations to stimulate muscle protein synthesis, but only in the presence of adequate amounts of amino acids (6,11,14–15). Given the inhibitory effect of insulin on protein breakdown, it is clear that unless amino acids are available, synthesis is unable to continue beyond the initial short term. Research on rat muscle has identified a number of potential cell signaling pathways (e.g., the activation of mTOR) that result in the initiation of the translation step of protein synthesis in response to insulin (6). Although hormones other than insulin (testosterone, cortisol, and possibly GH via IGF-1) may affect the responses of muscle protein turnover to feeding, their physiological importance relative to insulin during feeding is somewhat minor unless deficiencies or excess of these hormones occur. In other situations in which testosterone or anabolic steroids are elevated they can significantly stimulate muscle protein synthesis to promote muscle anabolism. Interestingly, we have recently shown that the hormone androstenedione (an over-the-counter hormone purported to increase testosterone levels) does not increase plasma testosterone concentrations and does not stimulate muscle protein synthesis in men (3,13).
NUTRITIONAL SUPPLEMENTATION AFTER RESISTANCE EXERCISE
Figure 3 summarizes our current understanding of how resistance exercise and feeding interact to bring about exercise-induced gains in muscle mass. When amino acids alone are administered after exercise, muscle protein synthesis increases more than with either exercise or amino acids alone. The provision of amino acids and carbohydrate (to stimulate insulin secretion) after resistance exercise exerts an even larger increase of muscle protein synthesis and net anabolism than any of the other stimuli. Figure 4 shows the progressive effect of exercise, amino acids alone at rest, exercise + amino acids alone, amino acids + carbohydrate at rest, and exercise + amino acids + carbohydrate on muscle protein synthesis and breakdown as compared with the basal period (rest + fasting) using stable isotope techniques with a three-compartment model. The studies labeled as #3, #4, and #5 in Figure 4 provided the nutrients in small boluses over a 3-h period, whereas study #6 gave the subjects all of the amino acids and carbohydrates in one bolus ingestion. As expected, the net balance (NB = synthesis − breakdown) of phenylalanine across the leg is negative after an overnight fast, an index of net catabolism (Study #1, Fig. 4); however it becomes less negative 3-h after a single bout of resistance exercise (Study #2, Fig. 4), but does not reach a positive value. On the other hand, a positive net balance, an index of net anabolism, is achieved when mixed (essential + nonessential) amino acids (40 g) are supplied at rest (Study #3, Fig. 4), and it is further amplified when the amino acids are given after exercise, in what appears to be a synergistic effect (Study #4, Fig. 4). When 40 g of mixed amino acids are ingested with 40 g of carbohydrate after an overnight fast, the rate of muscle protein synthesis is greater (Study #5, Fig. 4) than the combination of exercise and amino acids (Study #4, Fig. 4). This is most likely due to the inhibitory effect of insulin on muscle protein breakdown, and probably by the dual activation of intracellular signaling pathways by insulin and amino acids. Finally, a small bolus of essential amino acids (6 g) combined with 35 g of carbohydrate after one bout of resistance exercise resulted in the largest, and presumably synergistic, increase in mixed muscle protein synthesis and net anabolism (Study #6, Fig. 4).
We have also studied the most optimal time for nutritional provision in relation to the resistance exercise bout (12). It appears that consuming an essential amino acid/carbohydrate supplement 1 or 3 h after resistance exercise significantly increases amino acid availability, insulin concentrations, and muscle protein synthesis at a time when the synthetic machinery of the muscle cell is already activated (see Fig. 2). More recently, we have also shown that consuming the same supplement immediately before the resistance exercise bout is more anabolic than consuming the supplement immediately after exercise (15). In this situation, providing amino acids before exercise resulted in a greater delivery of amino acids to the working muscle during the exercise bout, which may have promoted amino acid uptake and attenuated breakdown. The lesser response of consuming the supplement immediately after exercise could be attributed to the potential delayed response for activation of translation initiation. It is not known what the effect of providing the supplement would be at other time points of elevated protein synthesis as shown in Figure 2 (i.e., 24 h postexercise), but one would expect the mechanisms for an increase in muscle protein synthesis to include enhanced activation of not only translation but also transcription.
Resistance exercise, essential amino acids, and insulin independently stimulate muscle protein synthesis via activation of various components of the translational apparatus. The enhanced muscle protein synthesis reported after a combination of all three is probably a synergistic effect of these components. Data from human muscle regarding the cellular mechanisms that regulate translation, transcription, and proteolysis are limited. Future research is needed to more accurately elucidate the pathways involved and how these pathways function in human muscle.
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